It is well understood in the art that bacterial infections may lead to formation of biofilms that protect the bacteria from the immune system and lead them to enter a quiescent, slow growth state that makes them resistant to most antibiotics (Donlan, R. M., et al., Clin. Microbiol. Rev. (2002) 15:167-193). The result is persistent, recurrent infections that are very difficult to eliminate. These biofilms include as a major component branched extracellular DNA molecules, whose key role was established by showing that DNAse treatment reduced biofilms (Whitchurch, C. B., et al., Science (2002) 295:1487; Petersen, F. C., et al., J. Bacteriol. (2004) 186:6327). The higher order meshwork structure of the DNA molecules is achieved by specific proteins generally designated DNABII proteins, with homologs found in most bacterial species, including proteins designated as IHF (integration host factor) and HU (histone like protein) (Swinger, K. K., et al., Curr. Opin. Struct. Biol. (2004) 14:28-35; Goodman, S. D., et al., Mucosal Immunity (2011) 4:625-637). The substantial homology of these proteins facilitates the cooperative formation of biofilms, a feature that further renders the bacteria problematic from a treatment perspective. Members of this class are known to be present in the extracellular environment (Winters, B. D., et al., Infect. Immun. (1993) 61:3259-3264; Lunsford, R. D., et al., Curr. Microbiol. (1996) 32:95-100; Kim, N., et al., J. Bacteriol. (2002) 184:6155-6162) and are known to force or stabilize bends in DNA, a key feature underlying higher order structure in other contexts (Teter, B., et al., Plasmid (2000) 43:73-84). Mutation of the ihfA gene in E. coli reduced or eliminated biofilm in vitro (Garcia-Contreras, R. (2008) PLoS ONE 3:e2394). The present invention is based on the concept that supplying a binding moiety with sufficiently high affinity for this class of proteins will extract the proteins from the biofilm and thereby provide an effective method of destroying the biofilm by destroying the ability of the protein to bind and hold together the branched DNA. A supplied binding moiety against the DNABII protein may also destroy its ability to bind to other components present in the biofilm.
The binding moieties, of which monoclonal antibodies or fragments thereof are an important embodiment, can be supplied directly to established biofilms or used to coat surfaces to provide an immuno-adsorbent for confining the DNABII protein(s) and thereby suppressing biofilm formation. Applications include treatments of bacterial infections by systemic administration, subcutaneous, topical or inhaled administration, as well as reduction of biofouling that affects pipelines and other industrial equipment. Application to corresponding biofilm associated diseases of animals is also part of the present invention.
PCT publication WO2011/123396 provides an extensive discussion of biofilms and provides for their removal by administering to a subject polypeptides that represent the DNABII protein itself as a whole or in part, thus causing the organism to generate antibodies that can destroy the integrity of the biofilm. This document also provides, in the alternative, supplying the antibodies themselves, either ex vivo to biofilms that exist outside an organism or to a subject to confer passive protection. Antibodies to other biofilm associated proteins have similarly been used to interfere with biofilms (Sun, D., et al., Clin. Diagn. Lab. Immunol. (2005) 12:93-100; Shahrooei, M., et al., Infect. Immun. (2009) 77:3670-3678; Novotny, L. A., et al., Vaccine (2009) 28:279-289).
The WO2011/123396 PCT application describes the use of polyclonal and monoclonal antibodies generated against a particular DNABII protein (E. coli integration host factor (IHF)) to treat an animal model of the common ear infection (otitis media) and an animal model for periodontal disease. It also describes generating active immunity by providing the protein, or peptides representing the protein to a subject. The present invention provides improved agents for passive immunity. The epitopes for two such monoclonal antibodies have been identified at the level of individual amino acids and are disclosed herein. One of these antibodies, TRL1068, was disclosed by the present applicants in PCT application US2014/057771. The non-identical but overlapping epitopes identify a region of the protein that is conformational with regard to the linear sequence of the IHF/HU protein, and thereby identifies favorable conformational features for an immunogen intended to generate an immune response with efficacy for interfering with a biofilm. Identification of the conformational nature of the epitope is also useful in the design of screening reagents for discovery of monoclonal antibodies or other homogeneous agents with biofilm disrupting activity and for affinity purification of such agents.